Detection of nucleic acid sequences (e.g., DNA sequences) is widely used in the fields of molecular biology and diagnosis for detection and identification of infectious diseases, genetic disorders and other research purposes. For detecting and analyzing a small quantity of nucleic acids, nucleic acid amplification technologies are used. Among them, the polymerase chain reaction (PCR) is the most widely used method.
While a very powerful technique, PCR has certain limitations as it requires multiple reiterative thermal cycles among different temperatures for different stages, e.g., denaturing, extension, and re-annealing respectively. Since each stage must be sufficiently long, the entire PCR reaction is very time consuming. To address this issue, the time duration for each stage can be limited. Yet, during the reiterative thermal cycling, a target sequence is extended and amplified efficiently only at the extension stage. As a result, PCR is limited in the size of the sequence to be amplified and the efficient amplification range is generally below 2000 bp. Furthermore, relying on precise cycling of a reaction cocktail between different temperatures, PCR requires expensive equipment such as a thermocycler. In addition, the repeated denaturing stage exposes the reaction cocktail to a temperature as high as 90° C. or above. Such a high temperature can damage some components of the cocktail and thereby negatively impact the length and quality of the amplified products. Thus, although there are more than 50 different PCR techniques in use, PCR-based detection methods remain expensive, time-consuming, instrument and reagent-intensive, and require extensive sample preparation.
An alternative to PCR is isothermal amplification. This alternative technology does not rely on reiterative cycles among different temperatures to achieve amplification and is therefore referred to as isothermal amplification. Several isothermal amplification techniques are known in the art. In general, isothermal amplification systems provide the advantages of speed, ease of use, the ability to utilize highly processive DNA polymerases, and do not require expensive thermocyclers. Yet, one of the limitations of isothermal amplification schemes is the relatively low specificity due to annealing of primers at temperatures lower (e.g., 37° C. or lower) as compared to those used in PCR. Thus, there is a need for highly specific isothermal amplification methods.
The CRISPR/CAS system is a class of nucleic acid targeting system originally discovered in prokaryotes that somewhat resemble siRNA/miRNA systems found in eukaryotes. The system consists of an array of short repeats with intervening variable sequences of constant length (i.e., clusters of regularly interspaced short palindromic repeats, or CRISPRs) and CRISPR-associated (CAS) proteins.
In CRISPR, each repetition contains a series of base pairs followed by the same or a similar series in reverse and then by 30 or so base pairs known as “spacer DNA.” The spacers are short segments of DNA from a virus, which have been removed from the virus or plasmid and incorporated into the host genome between the short repeat sequences, and serve as a “memory” of past exposures. The RNA of the transcribed CRISPR arrays is processed by a subset of the CAS proteins into small guide RNAs containing the viral or plasmid sequences, which direct CAS-mediated cleavage of viral or plasmid nucleic acid sequences that contain so-called protospacer adjacent motif (PAM) site and correspond to the small guide RNAs. That is, the CRISPR/CAS system functions as a prokaryotic immune system, as the spacers recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms thereby conferring resistance to exogenous genetic elements such as plasmids and phages.